Tag Archives: nanotoxicity

Scientists have come to a better understanding of the mechanism affecting silver nanoparticle toxicity according to an Aug. 30, 2016 news item on Nanowerk (Note: A link has been removed),

A senior fellow at the Faculty of Chemistry, MSU (Lomonosov Moscow State University), Vladimir Bochenkov together with his colleagues from Denmark succeeded in deciphering the mechanism of interaction of silver nanoparticles with the cells of the immune system. The study is published in the journal Nature Communications (“Dynamic protein coronas revealed as a modulator of silver nanoparticle sulphidation in vitro”).

‘Currently, a large number of products are containing silver nanoparticles: antibacterial drugs, toothpaste, polishes, paints, filters, packaging, medical and textile items. The functioning of these products lies in the capacity of silver to dissolve under oxidation and form ions Ag+ with germicidal properties. At the same time there are research data in vitro, showing the silver nanoparticles toxicity for various organs, including the liver, brain and lungs. In this regard, it is essential to study the processes occurring with silver nanoparticles in biological environments, and the factors affecting their toxicity,’ says Vladimir Bochenkov.

Caption: Increased intensity of the electric field near the silver nanoparticle surface in the excitation of plasmon resonance. Credit: Vladimir Bochenkov

The study is devoted to the protein corona — a layer of adsorbed protein molecules, which is formed on the surface of the silver nanoparticles during their contact with the biological environment, for example in blood. Protein crown masks nanoparticles and largely determines their fate: the speed of the elimination from the body, the ability to penetrate to a particular cell type, the distribution between the organs, etc.

According to the latest research, the protein corona consists of two layers: a rigid hard corona — protein molecules tightly bound with silver nanoparticles, and soft corona, consisting of weakly bound protein molecules in a dynamic equilibrium with the solution. Hitherto soft corona has been studied very little because of the experimental difficulties: the weakly bound nanoparticles separated from the protein solution easily desorbed (leave a particle remaining in the solution), leaving only the rigid corona on the nanoparticle surface.

The size of the studied silver nanoparticles was of 50-88 nm, and the diameter of the proteins that made up the crown — 3-7 nm. Scientists managed to study the silver nanoparticles with the protein corona in situ, not removing them from the biological environment. Due to the localized surface plasmon resonance used for probing the environment near the surface of the silver nanoparticles, the functions of the soft corona have been primarily investigated.

‘In the work we showed that the corona may affect the ability of the nanoparticles to dissolve to silver cations Ag+, which determine the toxic effect. In the absence of a soft corona (quickly sharing the medium protein layer with the environment) silver cations are associated with the sulfur-containing amino acids in serum medium, particularly cysteine and methionine, and precipitate as nanocrystals Ag2S in the hard corona,’ says Vladimir Bochenkov.

Ag2S (silver sulfide) famously easily forms on the silver surface even on the air in the presence of the hydrogen sulfide traces. Sulfur is also part of many biomolecules contained in the body, provoking the silver to react and be converted into sulfide. Forming of the nano-crystals Ag2S due to low solubility reduces the bioavailability of the Ag+ ions, reducing the toxicity of silver nanoparticles to null. With a sufficient amount of amino acid sulfur sources available for reaction, all the potentially toxic silver is converted into the nontoxic insoluble sulfide. Scientists have shown that what happens in the absence of a soft corona.

In the presence of a soft corona, the Ag2S silver sulfide nanocrystals are formed in smaller quantities or not formed at all. Scientists attribute this to the fact that the weakly bound protein molecules transfer the Ag+ ions from nanoparticles into the solution, thereby leaving the sulfide not crystallized. Thus, the soft corona proteins are ‘vehicles’ for the silver ions.

This effect, scientists believe, be taken into account when analyzing the stability of silver nanoparticles in a protein environment, and in interpreting the results of the toxicity studies. Studies of the cells viability of the immune system (J774 murine line macrophages) confirmed the reduction in cell toxicity of silver nanoparticles at the sulfidation (in the absence of a soft corona).

Vladimir Bochenkov’s challenge was to simulate the plasmon resonance spectra of the studied systems and to create the theoretical model that allowed quantitative determination of silver sulfide content in situ around nanoparticles, following the change in the absorption bands in the experimental spectra. Since the frequency of the plasmon resonance is sensitive to a change in dielectric constant near the nanoparticle surface, changes in the absorption spectra contain information about the amount of silver sulfide formed.

Knowledge of the mechanisms of formation and dynamics of the behavior of the protein corona, information about its composition and structure are extremely important for understanding the toxicity and hazards of nanoparticles for the human body. In prospect the protein corona formation can be used to deliver drugs in the body, including the treatment of cancer. For this purpose it will be enough to pick such a content of the protein corona, which enables silver nanoparticles penetrate only in the cancer cell and kill it.

Here’s a link to and a citation for the paper describing this fascinating work,

The process of moving on from testing on animals is laborious as new techniques are pioneered and, perhaps more arduously, people’s opinions and habits are changed. The People for the Ethical Treatment of Animals (PETA) organization focusing the research end of things has announced a means of predicting carbon nanotube toxicity in lungs according to an April 25, 2016 news item on Nanowerk (Note: A link has been removed),

A workshop organized last year [2015] by the PETA International Science Consortium Ltd has resulted in an article published today in the journal Particle and Fibre Toxicology (“Aerosol generation and characterization of multi-walled carbon nanotubes [MWCNTs] exposed to cells cultured at the air-liquid interface”). It describes aerosol generation and exposure tools that can be used to predict toxicity in human lungs following inhalation of nanomaterials.

Nanomaterials are increasingly being used in consumer products such as paints, construction materials, and food packaging, making human exposure to these materials more likely. One of the common ways humans may be exposed to these substances is by inhalation, therefore, regulatory agencies often require the toxicity of these materials on the lungs to be tested. These tests usually involve confining rats to small tubes the size of their bodies and forcing them to breathe potentially toxic substances before they are killed. However, time, cost, scientific and ethical issues have led scientists to develop methods that do not use animals. The tools described in the new article are used to deposit nanomaterials (or other inhalable substances) onto human lung cells grown in a petri dish.

Co-authors of the Particle and Fibre Toxicology article are scientists from the PETA Science Consortium , The Dow Chemical Company, Baylor University, and the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM).

“Promoting non-animal methods to assess nanotoxicity has been a focus of the PETA International Science Consortium”, said Dr. Monita Sharma, co-author of the publication and Nanotechnology Specialist at the Consortium, “we organized an international workshop last year on inhalation testing of nanomaterials and this review describes some of the tools that can be used to provide a better understanding of what happens in humans after inhaling these substances.” During the workshop, experts provided recommendations on the design of an in vitro test to assess the toxicity of nanomaterials (especially multi-walled carbon nanotubes) in the lung, including cell types, endpoints, exposure systems, and dosimetry considerations. Additional publications summarizing the outcomes of the workshop are forthcoming.

Because they are lawyers, I was intrigued by a Nov. 4, 2015 article on managing nanotechnology risks by Michael Lisak and James Mizgala of Sidley Austin LLP for Industry Week. I was also intrigued by the language (Note: A link has been removed),

The inclusion of nanotechnologies within manufacturing processes and products has increased exponentially over the past decade. Fortune recently noted that nanotechnology touches almost all Fortune 500 companies and that the industry’s $20 billion worldwide size is expected to double over the next decade. [emphasis mine]

Yet, potential safety issues have been raised and regulatory uncertainties persist. As such, proactive manufacturers seeking to protect their employees, consumers, the environment and their businesses – while continuing to develop, manufacture and market their products – may face difficult choices in how to best navigate this challenging and fluid landscape, while avoiding potential “nanotort,” [emphasis mine] whistleblower, consumer fraud and regulatory enforcement lawsuits. Doing so requires forward-thinking advice based upon detailed analyses of each manufacturer’s products and conduct in the context of rapidly evolving scientific, regulatory and legal developments.

I wonder how many terms lawyers are going to coin in addition to “nanotort”?

The lawyers focus largely on two types of nanoparticles, carbon nanotubes, with a special emphasis on multi-walled carbon nantubes (MWCNT) and nano titanium dioxide,

Despite this scientific uncertainty, international organizations, such as the International Agency for Research on Cancer [a World Health Organization agency], have already concluded that nano titanium dioxide in its powder form and multi-walled carbon nanotube-7 (“MWCNT-7”) [emphasis mine] are “possibly carcinogenic to humans.” As such, California’s Department of Public Health lists titanium dioxide and MWCNT-7 as “ingredients known or suspected to cause cancer, birth defects, or other reproductive toxicity as determined by the authoritative scientific bodies.” Considering that processed (i.e., non-powdered) titanium dioxide is found in products like toothpaste, shampoo, chewing gum and candies, it is not surprising that some have focused upon such statements.

There’s a lot of poison in the world, for example, apples contain seeds which have arsenic in them and, for another, peanuts can be carcinogenic and they can also kill you, as they are poison to people who are allergic to them.

On the occasion of Dunkin’ Donuts removing nano titanium dioxide as an ingredient in the powdered sugar used to coat donuts, I wrote a March 13, 2015 posting, where I quote extensively from Dr. Andrew Maynard’s (then director of the University of Michigan Risk Science Center now director of the Risk Innovation Lab at Arizona State University) 2020 science blog posting about nano titanium dioxide and Dunkin’ Donuts,

He describes some of the research on nano titanium dioxide (Note: Links have been removed),

… In 2004 the European Food Safety Agency carried out a comprehensive safety review of the material. After considering the available evidence on the same materials that are currently being used in products like Dunkin’ Donuts, the review panel concluded that there no evidence for safety concerns.

Most research on titanium dioxide nanoparticles has been carried out on ones that are inhaled, not ones we eat. Yet nanoparticles in the gut are a very different proposition to those that are breathed in.

Studies into the impacts of ingested nanoparticles are still in their infancy, and more research is definitely needed. Early indications are that the gastrointestinal tract is pretty good at handling small quantities of these fine particles. This stands to reason given the naturally occurring nanoparticles we inadvertently eat every day, from charred foods and soil residue on veggies and salad, to more esoteric products such as clay-baked potatoes. There’s even evidence that nanoparticles occur naturally inside the gastrointestinal tract.

You can find Andrew’s entire discussion in his March 12, 2015 post on the 2020 Science blog. Andrew had written earlier in a July 12, 2014 posting about something he terms ‘nano donut math’ as reported by As You Sow, the activist group that made a Dunkin’ Donuts shareholder proposal which resulted in the company’s decision to stop using nano titanium dioxide in the powdered sugar found on their donuts. In any event, Andrew made this point,

In other words, if a Dunkin’ Donut Powdered Cake Donut contained 8.9 mg of TiO2 particles smaller than 10 nm, it would have to have been doused with over 1 million tons of sugar coating! (Note update at the end of this piece)

Clearly something’s wrong here – either Dunkin’ Donuts are not using food grade TiO2 but a nanopowder with particle so small they would be no use whatsoever in the sugar coating (as well as being incredibly expensive, and not FDA approved). Or there’s something rather wrong with the analysis!

If it’s the latter – and it’s hard to imagine any other plausible reason for the data – it looks like As You Sow ended up using rather dubious figures to back up their stakeholder resolution. I’d certainly be interested in more information on the procedures Analytical Sciences used and the checks and balances they had in place, especially as there are a number of things that can mess up a particle analysis like this.

…

Update July 14: My bad, I made a slight error in the size distribution calculation first time round. This has been corrected in the article above. Originally, I cited the estimated Mass Median Diameter (MMD) of the TiO2 particles as 167 nm, and the Geometric Standard Deviation (GSD) as 1.6. Correcting an error in the Excel spreadsheet used to calculate the distribution (these things happen!) led to a revised estimate of MMD = 168 nm and a GSD of 1.44. These may look like subtle differences, but when calculating the estimated particle mass below 10 nm, they make a massive difference. With the revised figures, you’d expect less than one trillionth of a percent of the mass of the TiO2 powder to be below 10 nm!! (the original estimate was a tenth of a millionth of a percent). In other words – pretty much nothing! The full analysis can be found here.

Update November 16 2014. Based on this post, As You Sow checked the data from Analytical Sciences LLC and revised the report accordingly. This is noted above.

It would seem that if there are concerns over nano titanium dioxide in food, the biggest would not be the amounts ingested by consumers but inhalation by workers should they breathe in large quantities when they are handling the material.

As for the MWCNTs, they have long raised alarms but most especially the long MWCNTs and for people handling them during the course of their work day. Any MWCNTs found in sports equipment and other consumer products are bound in the material and don’t pose any danger of being inhaled into the lungs, unless they should be released from their bound state (e.g. fire might release them).

After some searching for MWCNT-7, I found something which seems also to be known as Mitsui MWCNT-7 or Mitsui 7-MWCNT (here’s one of my sources). As best I understand it, Mitsui is a company that produces an MWCNT which they have coined an MWCNT-7 and which has been used in nanotoxicity testing. As best I can tell, MWCNT is MWCNT-7.

The lawyers (Lisak and Mizgala) note things have changed for manufacturers since the early days and they make some suggestions,

…

One thing is certain – gone are the days when “sophisticated” manufacturers incorporating nanotechnologies within their products can reasonably expect to shield themselves by pointing to scientific and regulatory uncertainties, especially given the amount of money they are spending on research and development, as well as sales and marketing efforts.

First, manufacturers should fully understand the life-cycle of nanomaterials within their organization. For some, nanomaterials may be an explicit focus of innovation and production, making it easier to pinpoint where nanotechnology fits into their processes and products. For others, nanomaterials may exist either higher-up or in the back-end of their products’ supply chain. …

Second, manufacturers should understand and stay current with the scientific state-of-the-art as well as regulatory requirements and developments potentially applicable to their employees, consumers and the environment. An important consideration related to efforts to understand the state-of-the-art is whether or not manufacturers should themselves expend resources to advance “the science” in seeking to help find answers to some of the aforementioned uncertainties. …

The lawyers go on to suggest that manufacturers should consider proactively researching nanotoxicity so as to better defend themselves against any future legal suits.

Encouraging companies to proactive with toxicity issues is in line with what seems to be an international (Europe & US) regulatory movement putting more onus on producers and manufacturers to take responsibility for safety testing. (This was communicated to me in a conversation I had with an official at the European Union Joint Research Centre where he mentioned REACH regulations and the new emphasis in response to my mention of similar FDA (US Food and Drug Administration) regulations. (We were at the 2014 9th World Congress on Alternatives to Animal Testing in Prague, Czech republic.)

For anyone interested in the International Agency for Research on Cancer you can find it here.

An Oct. 8, 2015 news item on Nanowerk offers some context for why researchers at the University of California at Los Angeles (UCLA) are studying silver nanoparticles and their entry into the water system,

More than 2,000 consumer products today contain nanoparticles — particles so small that they are measured in billionths of a meter.

Manufacturers use nanoparticles to help sunscreen work better against the sun’s rays and to make athletic apparel better at wicking moisture away from the body, among many other purposes.

Of those products, 462 — ranging from toothpaste to yoga mats — contain nanoparticles made from silver, which are used for their ability to kill bacteria. But that benefit might be coming at a cost to the environment. In many cases, simply using the products as intended causes silver nanoparticles to wind up in rivers and other bodies of water, where they can be ingested by fish and interact with other marine life.

For scientists, a key question has been to what extent organisms retain those particles and what effects they might have.

I’d like to know where they got those numbers “… 2,000 consumer products …” and “… 462 — ranging from toothpaste to yoga mats — contain nanoparticles made from silver… .”

A new study by the University of California Center for Environmental Implications of Nanotechnology has found that smaller silver nanoparticles were more likely to enter fish’s bodies, and that they persisted longer than larger silver nanoparticles or fluid silver nitrate. The study, published online in the journal ACS Nano, was led by UCLA postdoctoral scholars Olivia Osborne and Sijie Lin, and Andre Nel, director of UCLA’s Center for Environmental Implications of Nanotechnology and associate director of the California NanoSystems Institute at UCLA.

Nel said that although it is not yet known whether silver nanoparticles are harmful, the research team wanted to first identify whether they were even being absorbed by fish. CEIN, which is funded by the National Science Foundation, is focused on studying the effects of nanotechnology on the environment.

In the study, researchers placed zebrafish in water that contained fluid silver nitrate and two sizes of silver nanoparticles — some measuring 20 nanometers in diameter and others 110 nanometers. Although the difference in size between these two particles is so minute that it can only be seen using high-powered transmission electron microscopes, the researchers found that the two sizes of particles affected the fish very differently.

The researchers used zebrafish in the study because they have some genetic similarities to humans, their embryos and larvae are transparent (which makes them easier to observe). In addition, they tend to absorb chemicals and other substances from water.

Osborne said the team focused its research on the fish’s gills and intestines because they are the organs most susceptible to silver exposure.

“The gills showed a significantly higher silver content for the 20-nanometer than the 110-nanometer particles, while the values were more similar in the intestines,” she said, adding that both sizes of the silver particles were retained in the intestines even after the fish spent seven days in clean water. “The most interesting revelation was that the difference in size of only 90 nanometers made such a striking difference in the particles’ demeanor in the gills and intestines.”

The experiment was one of the most comprehensive in vivo studies to date on silver nanoparticles, as well as the first to compare silver nanoparticle toxicity by extent of organ penetration and duration with different-sized particles, and the first to demonstrate a mechanism for the differences.

Osborne said the results seem to indicate that smaller particles penetrated deeper into the fishes’ organs and stayed there longer because they dissolve faster than the larger particles and are more readily absorbed by the fish.

Lin said the results indicate that companies using silver nanoparticles have to strike a balance that recognizes their benefits and their potential as a pollutant. Using slightly larger nanoparticles might help make them somewhat safer, for example, but it also might make the products in which they’re used less effective.

He added that data from the study could be translated to understand how other nanoparticles could be used in more environmentally sustainable ways.

Nel said the team’s next step is to determine whether silver particles are potentially harmful. “Our research will continue in earnest to determine what the long-term effects of this exposure can be,” he said.

This grant seems to have gotten its start at a workshop held at the US Environmental Protection Agency (EPA) in Washington, D.C., Feb. 24-25, 2015 as per this webpage on the People for Ethical Treatment of Animals (PETA) International Science Consortium Limited website,

The invitation-only workshop included experts from different sectors (government, industry, academia and NGO) and disciplines (in vitro and in vivo inhalation studies of NMs, fibrosis, dosimetry, fluidic models, aerosol engineering, and regulatory assessment). It focused on the technical details for the development and preliminary assessment of the relevance and reliability of an in vitro test to predict the development of pulmonary fibrosis in cells co-cultured at the air-liquid interface following exposure to aerosolized multi-walled carbon nanotubes (MWCNTs). During the workshop, experts made recommendations on cell types, exposure systems, endpoints and dosimetry considerations required to develop thein vitro model for hazard identification of MWCNTs.

The method is intended to be included in a non-animal test battery to reduce and eventually replace the use of animals in studies to assess the inhalation toxicity of engineered NMs. The long-term vision is to develop a battery of in silico and in vitro assays that can be used in an integrated testing strategy, providing comprehensive information on biological endpoints relevant to inhalation exposure to NMs which could be used in the hazard ranking of substances in the risk assessment process.

The PETA International Science Consortium Ltd. announced today the winners of a $200,000 award for the design of an in vitro test to predict the development of lung fibrosis in humans following exposure to nanomaterials, such as multi-walled carbon nanotubes.

Professor Dr. Barbara Rothen-Rutishauser of the Adolphe Merkle Institute at the University of Fribourg, Switzerland and Professor Dr. Vicki Stone of the School of Life Sciences at Heriot-Watt University, Edinburgh, U.K. will jointly develop the test method. Professor Rothen-Rutishauser co-chairs the BioNanomaterials research group at the Adolphe Merkle Institute, where her research is focused on the study of nanomaterial-cell interactions in the lung using three-dimensional cell models. Professor Vicki Stone is the Director of the Nano Safety Research Group at Heriot-Watt University and the Director of Toxicology for SAFENANO.

The Science Consortium is also funding MatTek Corporation for the development of a three-dimensional reconstructed primary human lung tissue model to be used in Professors Rothen-Rutishauser and Stone’s work. MatTek Corporation has extensive expertise in manufacturing human cell-based, organotypic in vitro models for use in regulatory and basic research applications. The work at MatTek will be led by Dr. Patrick Hayden, Vice President of Scientific Affairs, and Dr. Anna Maione, head of MatTek’s airway models research group.

I was curious about MatTek Corporation and found this on company’s About Us webpage,

MatTek Corporation was founded in 1985 by two chemical engineering professors from MIT. In 1991 the company leveraged its core polymer surface modification technology into the emerging tissue engineering market.

MatTek Corporation is at the forefront of tissue engineering and is a world leader in the production of innovative 3D reconstructed human tissue models. Our skin, ocular, and respiratory tissue models are used in regulatory toxicology (OECD, EU guidelines) and address toxicology and efficacy concerns throughout the cosmetics, chemical, pharmaceutical and household product industries.

EpiDerm™, MatTek’s first 3D human cell based in vitro model, was introduced in 1993 and became an immediate technical and commercial success.

I wish them good luck in their research on developing better ways to test toxicity.

There is a puzzling and exciting announcement from the Canadian Light Source in a May 27, 2015 news item on Nanowerk,

Precious metals like silver and gold have biomedical properties that have been used for centuries, but how do these materials effectively combat the likes of cancer and bacteria without contaminating the patient and the environment?

These are the questions that researchers from Dalhousie University and the Canadian Light Source are trying to find out.

Perhaps I’m misreading the announcement but the statement that nanosilver and nanogold don’t contaminate the patient or the environment is a bit exuberant. There are published studies examining questions about whether or not nanosilver may affect the environment and health and the answer is that no one is certain yet. You can read more about two studies highlighted in my February 28, 2013 posting titled: Silver nanoparticles, water, the environment, and toxicity. As for nanosilver and nanogold not contaminating patients, that too is a problematic statement. For example, I have this paper which cites several studies on nanogold and possible toxicity. The paper itself is a plea to standardize testing and protocols so researchers can do a better job of establishing toxicity issues with nanogold.

Reservations aside, it’s good to learn of some Canadian research in this area. From a May 26, 2015 Canadian Light Source news release, which originated the news item, provides more details about the research and its current focus on nanosilver,

“Gold and silver are both exciting materials,” said Peng Zhang, Associate Professor of Chemistry at Dalhousie. “We can use gold to either detect or kill cancer cells. Silver is also excited and a very promising material as an antibacterial agents.”

Zhang said that if you compare silver to current antibiotics, silver does not show drug-resistant behaviour. “But with silver, so far, we are not finding that,” he added.

Finding out why silver is such a great antibacterial agent is the focus of Zhang’s research, recently published in the journal Langmuir.

“We want to understand the relationship between the atomic structure and bioactivity of nanosilver as to why it is so efficient at inhibiting bacterial activity. It’s a big puzzle.”

Zhang said it is very hard to understand what is happening at the atomic level. Using small nanosilver particles is the most effective way, because when you make silver small, you can expect higher activity because of the increased surface area.

This poses another problem however, as the nanosilver needs to be stabilized with a coating or the silver particles will bond together forming large pieces of silver that do not efficiently interact with the bacteria.

Zhang’s group used two different coatings to compare the effectiveness of the silver as an antibacterial agent. The first was a small amino acid coating and the other was a larger polymer coating. And after testing the interactions between the nanosilver and the bacteria, and looking at the atomic structure of nanosilver using the CLS and the Advanced Photon Source, the researchers were surprised to find that the thicker, larger polymer coating actually created a better delivery method for sliver to inhibit the bacteria.

“We proposed that the small amino acid coating would bind so tightly to the silver surface that it would be difficult for the silver atoms to interact with the bacteria, whereas the polymers are actually very good at staying in place and still releasing sufficient amount of silver with the bacteria.”

Zhang said the next steps will be to find out if the nanosilver is actually attacking good cells in living systems before they can make any further progress on determining whether nanosilver is an effective and efficient antibactieral agent that could be used to cure human and animal diseases.

Alicia Taylor, a graduate student at UC Riverside, surrounded by buckets of effluent from the septic tank system she used for her research. Courtesy: University of California at Riverside

Those buckets of efflluent are strangely compelling. I think it’s the abundance of orange. More seriously, a March 2, 2015 news item on Nanowerk poses a question about copper nanoparticles,

What do a human colon, septic tank, copper nanoparticles and zebrafish have in common?

They were the key components used by researchers at the University of California, Riverside and UCLA [University of California at Los Angeles] to study the impact copper nanoparticles, which are found in everything from paint to cosmetics, have on organisms inadvertently exposed to them.

The researchers found that the copper nanoparticles, when studied outside the septic tank, impacted zebrafish embryo hatching rates at concentrations as low as 0.5 parts per million. However, when the copper nanoparticles were released into the replica septic tank, which included liquids that simulated human digested food and household wastewater, they were not bioavailable and didn’t impact hatching rates.

“The results are encouraging because they show with a properly functioning septic tank we can eliminate the toxicity of these nanoparticles,” said Alicia Taylor, a graduate student working in the lab of Sharon Walker, a professor of chemical and environmental engineering at the University of California, Riverside’s Bourns College of Engineering.

…

The research comes at a time when products with nanoparticles are increasingly entering the marketplace. While the safety of workers and consumers exposed to nanoparticles has been studied, much less is known about the environmental implications of nanoparticles. The Environmental Protection Agency is currently accessing the possible effects of nanomaterials, including those made of copper, have on human health and ecosystem health.

The UC Riverside and UCLA [University of California at Los Angeles] researchers dosed the septic tank with micro copper and nano copper, which are elemental forms of copper but encompass different sizes and uses in products, and CuPRO, a nano copper-based material used as an antifungal agent to spray agricultural crops and lawns.

While these copper-based materials have beneficial purposes, inadvertent exposure to organisms such as fish or fish embryos has not received sufficient attention because it is difficult to model complicated exposure environments.

The UC Riverside researchers solved that problem by creating a unique experimental system that consists of the replica human colon and a replica two-compartment septic tank, which was originally an acyclic septic tank. The model colon is made of a custom-built 20-inch-long glass tube with a 2-inch diameter with a rubber stopper at both ends and a tube-shaped membrane typically used for dialysis treatments within the glass tube.

To simulate human feeding, 100 milliliters of a 20-ingredient mixture that replicated digested food was pumped into the dialysis tube at 9 a.m., 3 p.m. and 9 p.m. for five-day-long experiments over nine months.

The septic tank was filled with waste from the colon along with synthetic greywater, which is meant to simulate wastewater from sources such as sinks and bathtubs, and the copper nanoparticles. The researchers built a septic tank because 20 to 30 percent of American households rely on them for sewage treatment. Moreover, research has shown up to 40 percent of septic tanks don’t function properly. This is a concern if the copper materials are disrupting the function of the septic system, which would lead to untreated waste entering the soil and groundwater.

Once the primary chamber of the septic system was full, liquid began to enter the second chamber. Once a week, the effluent was drained from the secondary chamber and it was placed into sealed five-gallon containers. The effluent was then used in combination with zebrafish embryos in a high content screening process using multiwall plates to access hatching rates.

The remaining effluent has been saved and sits in 30 five-gallon buckets in a closet at UC Riverside because some collaborators have requested samples of the liquid for their experiments.

Caption: Low-cost experiments to test the toxicity of nanomaterials focused on populations of roundworms. Rice University scientists were able to test 20 nanomaterials in a short time, and see their method as a way to determine which nanomaterials should undergo more extensive testing. Credit: Zhong Lab/Rice University

Until now, ‘cute’ and ‘adorable’ are not words I would have associated with worms of any kind or with Rice University, for that matter. It’s amazing what a single image can do, eh?

A Feb. 3, 2015 news item on Azonano describes how roundworms have been used in research investigating the toxicity of various kinds of nanoparticles,

The lowly roundworm is the star of an ambitious Rice University project to measure the toxicity of nanoparticles.

The low-cost, high-throughput study by Rice scientists Weiwei Zhong and Qilin Li measures the effects of many types of nanoparticles not only on individual organisms but also on entire populations.

The Rice researchers tested 20 types of nanoparticles and determined that five, including the carbon-60 molecules (“buckyballs”) discovered at Rice in 1985, showed little to no toxicity.

Others were moderately or highly toxic to Caenorhabditis elegans, several generations of which the researchers observed to see the particles’ effects on their health.

The results were published by the American Chemical Society journal Environmental Sciences and Technology. They are also available on the researchers’ open-source website.

“Nanoparticles are basically new materials, and we don’t know much about what they will do to human health and the health of the ecosystem,” said Li, an associate professor of civil and environmental engineering and of materials science and nanoengineering. “There have been a lot of publications showing certain nanomaterials are more toxic than others. So before we make more products that incorporate these nanomaterials, it’s important that we understand we’re not putting anything toxic into the environment or into consumer products.

“The question is, How much cost can we bear?” she said. “It’s a long and expensive process to do a thorough toxicological study of any chemical, not just nanomaterials.” She said that due to the large variety of nanomaterials being produced at high speed and at such a large scale, there is “an urgent need for high-throughput screening techniques to prioritize which to study more extensively.”

Rice’s pilot study proves it is possible to gather a lot of toxicity data at low cost, said Zhong, an assistant professor of biosciences, who has performed extensive studies on C. elegans, particularly on their gene networks. Materials alone for each assay, including the worms and the bacteria they consumed and the culture media, cost about 50 cents, she said.

The researchers used four assays to see how worms react to nanoparticles: fitness, movement, growth and lifespan. The most sensitive assay of toxicity was fitness. In this test, the researchers mixed the nanoparticles in solutions with the bacteria that worms consume. Measuring how much bacteria they ate over time served as a measure of the worms’ “fitness.”

“If the worms’ health is affected by the nanoparticles, they reproduce less and eat less,” Zhong said. “In the fitness assay, we monitor the worms for a week. That is long enough for us to monitor toxicity effects accumulated through three generations of worms.” C. elegans has a life cycle of about three days, and since each can produce many offspring, a population that started at 50 would number more than 10,000 after a week. Such a large number of tested animals also enabled the fitness assay to be highly sensitive.

The researchers’ “QuantWorm” system allowed fast monitoring of worm fitness, movement, growth and lifespan. In fact, monitoring the worms was probably the least time-intensive part of the project. Each nanomaterial required specific preparation to make sure it was soluble and could be delivered to the worms along with the bacteria. The chemical properties of each nanomaterial also needed to be characterized in detail.

The researchers studied a representative sampling of three classes of nanoparticles: metal, metal oxides and carbon-based. “We did not do polymeric nanoparticles because the type of polymers you can possibly have is endless,” Li explained.

They examined the toxicity of each nanoparticle at four concentrations. Their results showed C-60 fullerenes, fullerol (a fullerene derivative), titanium dioxide, titanium dioxide-decorated nanotubes and cerium dioxide were the least damaging to worm populations.

They also determined the degree to which surface chemistry affected the toxicity of some particles. While amine-functionalized multiwalled nanotubes proved highly toxic, hydroxylated nanotubes had the least toxicity, with significant differences in fitness, body length and lifespan.

A complete and interactive toxicity chart for all of the tested materials is available online.

Zhong said the method could prove its worth as a rapid way for drug or other companies to narrow the range of nanoparticles they wish to put through more expensive, dedicated toxicology testing.

“Next, we hope to add environmental variables to the assays, for example, to mimic ultraviolet exposure or river water conditions in the solution to see how they affect toxicity,” she said. “We also want to study the biological mechanism by which some particles are toxic to worms.”

Here’s a citation for the paper and links to the paper and to the researchers’ website,

This is the first time I’ve seen testing of two organs for possible adverse effects from nanoparticles. In this case, the researchers were especially interested in the liver. From an Aug. 12, 2014 news item on Azonano,

Nanoparticles in food, sunscreen and other everyday products have many benefits. But Cornell [University] biomedical scientists are finding that at certain doses, the particles might cause human organ damage.

A recently published study in Lab on a Chip by the Royal Society of Chemistry and led by senior research associate Mandy Esch shows that nanoparticles injure liver cells when they are in microfluidic devices designed to mimic organs of the human body. The injury was worse when tested in two-organ systems, as opposed to single organs – potentially raising concerns for humans and animals.

Anne Ju’s Aug. 11, 2014 article for Cornell University’s Chronicle describes the motivation for this work and the research itself in more detail,

“We are looking at the effects of what are considered to be harmless nanoparticles in humans,” Esch said. “These particles are not necessarily lethal, but … are there other consequences? We’re looking at the non-lethal consequences.”

She used 50-nanometer carboxylated polystyrene nanoparticles, found in some animal food sources and considered model inert particles. Shuler’s lab specializes in “body-on-a-chip” microfluidics, which are engineered chips with carved compartments that contain cell cultures to represent the chemistry of individual organs.

In Esch’s experiment, she made a human intestinal compartment, a liver compartment and a compartment to represent surrounding tissues in the body. She then observed the effects of fluorescently labeled nanoparticles as they traveled through the system.

Esch found that both single nanoparticles as well as small clusters crossed the gastrointestinal barrier and reached liver cells, and the liver cells released an enzyme called aspartate transaminase, known to be released during cell death or damage.

It’s unclear exactly what damage is occurring or why, but the results indicate that the nanoparticles must be undergoing changes as they cross the gastrointestinal barrier, and that these alterations may change their toxic potential, Esch said. Long-term consequences for organs in proximity could be a concern, she said.

“The motivation behind this study was twofold: one, to show that multi-organ, in vitro systems give us more information when testing for the interaction of a substance with the human body, and two … to look at nanoparticles because they have a huge potential for medicine, yet adverse effects have not been studied in detail yet,” Esch said.

Mary Macleod’s July 3, 2014 article for Chemistry World features a diagram of the two-organ system and more technical details about the research,

Schematic of the two-organ system [downloaded from http://www.rsc.org/chemistryworld/2014/07/nanoparticle-liver-gastrointestinal-tract-microfluidic-chip]

HepG2/C3A cells were used to represent the liver, with the intestinal cell co-culture consisting of enterocytes (Caco-2) and mucin-producing (HT29-MTX) cells. Carboxylated polystyrene nanoparticles were fluorescently labelled so their movement between the chambers could be tracked. Levels of aspartate transaminase, a cytosolic enzyme released into the culture medium upon cell death, were measured to give an indication of liver damage.

The study saw that single nanoparticles and smaller nanoparticle aggregates were able to cross the GI barrier and reach the liver cells. The increased zeta potentials of these nanoparticles suggest that crossing the barrier may raise their toxic potential. However, larger nanoparticles, which interact with cell membranes and aggregate into clusters, were stopped much more effectively by the GI tract barrier.

The gastrointestinal tract is an important barrier preventing ingested substances crossing into systemic circulation. Initial results indicate that soluble mediators released upon low-level injury to liver cells may enhance the initial injury by damaging the cells which form the GI tract. These adverse effects were not seen in conventional single-organ tests.

While this research is deeply concerning, it should be noted the researchers are being very careful in their conclusions as per Ju’s article, “It’s unclear exactly what damage is occurring or why, but the results indicate that the nanoparticles must be undergoing changes as they cross the gastrointestinal barrier, and that these alterations may change their toxic potential … Long-term consequences for organs in proximity could be a concern … .”

The head for this post is meant to inform you that French language reading skills will be needed if you want to read the materials for the press kit about toxicity and nanomaterials that ANSES (France Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail; French Agency for Food, Environmental and Occupational Health & Safety; Comment: it seems perfect from a cultural perspective that the French would put food first in this list of agency responsibilities) released on May 15, 2014 as per the news item on Nanowerk (Note: A link has been removed),

Confronted with the wide range of nanomaterials in everyday life and the many questions surrounding them, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) published a review (pdf; in French; [the review is a press kit or dossier de presse]) of the available literature on health and environmental issues relating to manufactured nanomaterials. This will help clarify scientific understanding and demonstrate the toxic effects of some nanomaterials on living organisms and the environment. It also emphasises the complexity of understanding the various situations to which humans and the environment are exposed, and the limitations of existing risk assessment methodologies. Due to this complexity, it is difficult to assess the specific risks associated with nanomaterials. Given the time it would require, the Agency recommendsthe immediate implementation of tools to improve risk management through a stronger regulatory framework at European level.

The English language version of the ANSES May 15, 2014 news release, which originated the news item, provides some historical perspective on nanomaterials while describing how the review (press kit) supports the agency’s recommendations for the future,

Some “older” nanomaterials have been around for almost a century and certain nanoparticles can also be produced naturally. It was not however until the late 1990s that technological developments emerged that led to a wide diversification of industrial applications. Nanomaterials are now found in many everyday products: cosmetics, textiles, food, paints, medical applications, etc. This technological deployment has been accompanied by studies on their potential health impact, yet many doubts remain as to their effects on health and the environment. This uncertainty has led to questions about the degree to which these risks are controlled, and the appropriate regulatory framework.

…

Drawing on a review of all available data and scientific publications around the world, the report documents the effects identified on living organisms. Based on in vitro and in vivo animal tests, it first demonstrates the ability of nanomaterials to cross physiological barriers, and highlights the toxicity of certain nanomaterials, noting that there are currently no data directly concerning humans, due to the lack of epidemiological studies.

…

Regarding research, the Agency recommends implementing multidisciplinary projects to develop knowledge of the characteristics and hazards of nanomaterials, throughout the product life cycle. This mainly involves promoting the development of appropriate safety tests for assessing the health risks of products containing nanomaterials intended to be placed on the market.

In addition, ANSES is calling immediately for a strengthened regulatory framework for manufactured nanomaterials at the European level, in order to better characterise each substance and its uses, taking into account the entire product life cycle.

I haven’t had the time to read the report and may not in the future but I did look for a bibliography, which I did not find, hoping to discover what they meant when they said nanomaterials have been present for a century. Depending on how one views these things, one could say that nanomaterials have always been present on planet earth.

As for the timing of this announcement, one has to notice that it comes on the heels of an announcement from the European Union of a ‘public consultation on measures to increase transparency on nanomaterials on the market’ (see my earlier May 16, 2014 post*).

* Date corrected from May 15 to May 16 at 9:50 am PDT on May 16, 2014.